On Measuring miRNAs after Transient Transfection of Mimics ...€¦ · On Measuring miRNAs after...
Transcript of On Measuring miRNAs after Transient Transfection of Mimics ...€¦ · On Measuring miRNAs after...
On Measuring miRNAs after Transient Transfection ofMimics or Antisense InhibitorsDaniel W. Thomson1,2, Cameron P. Bracken1,2, Jan M. Szubert1, Gregory J. Goodall1,2,3*
1 Centre for Cancer Biology, South Australia Pathology, Adelaide, South Australia, Australia, 2 Department of Medicine, University of Adelaide, Adelaide, South Australia,
Australia, 3 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia
Abstract
The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespreaduse of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs(antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does notaccurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in manypublished reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNAin whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that thesupraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, becausethe majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionallyactive miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also thepotential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directlyinterfere with the PCR reaction used to measure miRNA level.
Citation: Thomson DW, Bracken CP, Szubert JM, Goodall GJ (2013) On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors. PLoSONE 8(1): e55214. doi:10.1371/journal.pone.0055214
Editor: Georg Stoecklin, German Cancer Research Center, Germany
Received November 7, 2012; Accepted December 19, 2012; Published January 24, 2013
Copyright: � 2013 Thomson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a PhD scholarship from Adelaide University (to DWT), a fellowship from the National Breast Cancer Foundation Australia(to CPB) and by grants from the National Health and Medical Research Council (to GJG and CPB). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
MicroRNAs are small endogenous RNA molecules that guide
the RNA-protein complex, RISC (RNA induced silencing
complex), to target sequences in mRNAs. The biosynthesis and
functions of miRNAs have been reviewed recently [1]. RISC-
loaded miRNAs bind in a sequence-specific manner to target
mRNAs, initiating their repression through a combination of
translational inhibition, RNA destabilisation (via de-capping and
de-adenylation) or, albeit rarely in mammals, direct RISC-
mediated mRNA cleavage [2,3,4,5,6,7]. The majority of mRNA
transcripts are subject to direct miRNA-mediated regulation,
largely via interactions with target 39 untranslated regions.
Consequently, miRNAs are directly or indirectly involved in most
biological processes and have been extensively implicated in such
areas as development, immune regulation and cancer progression.
Results and Discussion
For a miRNA to be functional, it must be incorporated into
RISC. While qPCR is a simple and commonly used method to
measure the level of a miRNA, it does not distinguish between
miRNAs in functional or non-functional pools. To assess whether
the majority of transiently transfected miRNA resides in a
functional location, we transfected miR-200a mimic into MDA-
MB-231 cells, which have very little endogenous miR-200a, and
measured the miR-200a level after 2 days by TaqMan qPCR
assay or by immunoprecipitation with anti-Ago antibody followed
by deep sequencing. Measurement of the transfected miRNA by
qPCR indicated miR-200a was increased by .1000- fold, to a
level vastly greater than the most abundant endogenous miRNAs,
such as miR-125b and miR-16 (Fig. 1). However, we found that
double-stranded miRNA mimics added to cell extracts post-lysis
were also detected at high level by the qPCR (Fig. 1), demon-
strating that qPCR amplification alone does not necessarily
indicate functionality.
To measure the level of functional miRNA in a manner that
avoids detecting miRNA mimic trapped in non-functional
locations, we immunoprecipitated UV cross-linked RISC from
control and transfected cells and measured the amount of RISC-
associated miR-200a by deep sequencing of the miRNA-sized
RNA fraction in the immunoprecipitate. This revealed that the
amount of RISC-associated miR-200a in the transfected cells was
approximately equal to the level of other abundant miRNAs
(Fig. 2A). This is proportionally much less than the level of miR-
200a measured by qPCR (Fig. 1, Fig. 2B), indicating most of the
transfected miRNA mimic is not bound to Argonaute and
consequently is not functional. Similar results were obtained
following transfection of a different miRNA, miR-200b (data not
shown). Thus, although qPCR is a valid technique to measure
total miRNA amount, this can be very different from the amount
of functional miRNA.
Given the majority of miRNA mimic detected by qPCR did not
represent the active Argonaute-bound population, we determined
its sub-cellular localisation by transfecting a fluorescent siRNA and
examining the transfected cells by fluorescence microscopy. The
PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e55214
majority of the siRNA did not co-localise with Argonaute (Fig. 3A;
Fig. 3B), which is consistent with earlier reports of transfected
siRNA localising in large cytoplasmic aggregates that are distinct
from the GW bodies that are known for their role in RNA
silencing [8]. Instead the vast majority of miRNA transfected with
either HiPerfect, (Fig. 4a), RNAi-Max (Fig. 4b) or Lipofectamine
2000 (data not shown) localised with or adjacent to lysosomes,
matching earlier reports of lipid-based siRNA transfection [9,10].
Therefore, the high level of transfected miRNA detected by qPCR
is largely attributable to their retention within vesicles and
subsequent amplification by qPCR following lysis. Hence, the
use of qPCR to measure a miRNA after transient transfection can
give the false impression that the miRNA is at massively non-
physiological level, whereas the amount of miRNA bound to
Argonaute may indeed be appropriately physiological. On the
other hand, it is conceivable that an inefficient transfection that
results in just a small proportion of cells being transfected could
appear to produce an adequate level of miRNA, if measured by
qPCR. It is more appropriate to use an assay of miRNA function
to verify the effectiveness of the transfection.
Of additional interest to users of miRNA mimics for transient
transfection, we were able to confirm from our sequencing of the
Argonaute-bound pool of small RNAs, that while a miRNA mimic
with unmodified passenger strand results in abundant incorpora-
tion of the passenger strand into RISC (Fig. 5A), raising the
potential for extensive off-target effects, a mimic that is modified to
limit the incorporation of the passenger strand into RISC does
indeed achieve this (Fig. 5B). Although the merits of modified
mimics have been previously recognised, published evidence for
this is limited to date and has been based largely on reporter assays
comparing the response of reporters that harbour a target site for
either the siRNA sense strand or passenger strand (http://
products.invitrogen.com/ivgn/product/AM17100). Our observa-
tion provides additional support for the lack of incorporation of
modified passenger strand.
qPCR is also sometimes used to verify the inhibition of a
miRNA by transiently transfected antisense inhibitor, but this can
also be problematic because the antisense inhibitor can directly
inhibit the qPCR reaction. For example, in an experiment where
transfection of miR-200a antisense inhibitor into MCF7 cells
produced an apparent ,50% decrease in miR-200a levels as
measured by qPCR (Fig. 6A), we found that much of the apparent
decrease in miRNA was attributable to the suppressive effect of
antisense inhibitor on the PCR reaction itself. This was revealed
by the addition of the same amount of antisense inhibitor directly
to the cells after lysis by TRIzol, but prior to RNA extraction,
which appeared to give a similar decrease in the level of miR-200a
as measured by qPCR. Coupled with the fact that most of the
transfected oligonucleotide is located in vesicles, this indicates that
the qPCR may be largely measuring the inhibitory effect of the
vesicle-associated antisense inhibitors on the qPCR, rather than its
antisense activities within cells. We note that both 29-O-Methyl
and LNA (locked nucleic acid) miRNA inhibitors are similarly
subject to this phenomenon (Fig. 6A, B). This complements
previous observations that the LNA:miRNA complex interferes
with the binding of the Northern blot probe when measuring
miRNA inhibition by Northern blot [11].
Whilst miRNA mimics and antisense inhibitors are valuable
tools, our observations indicate caveats to the analysis of miRNA
and antisense inhibitor transfection that are apparently not
universally appreciated, leading to the surprisingly frequent use
in the literature (examples available on request) of qPCR for
mRNA measurement when a readout of function would be more
appropriate. Better options are the use of a miRNA reporter (such
as luciferase or a fluorescent protein under the control of miRNA
target sites) to report the relative functional level of a miRNA, or
measurement of the miRNA level following Argonaute immuno-
precipitation.
Materials and Methods
Quantitative PCR following miRNA mimic transfection oraddition post lysis
60 nM miR-200a mimic (Ambion or Genepharma) was
transfected into MDA-MB-231 cells using Lipofectamine 2000
transfection reagent (Invitrogen) or Lipofectamine RNAiMAX
(Invitrogen) compared to a scrambled control miRNA. Cells were
incubated at 37uC, 5% CO2 for 48 h and harvested with TRIzol
reagent (Invitrogen). Prior to RNA extraction samples were doped
(addition without transfection) with the equivalent amounts of
miR-200a mimic. qRT-PCR was performed using MultiScribe
RT and TaqMan probes (Applied Biosystems) for hsa-miR-200a,
hsa-miR-16 and hsa-miR-125b. Unmodified miRNA mimics were
obtained from GenePharma and proprietary modified miRNAs
(designed for selective incorporation of the guide strand into
RISC) are from Ambion.
Argonaute: miRNA immunoprecipitationMDA-MB-231 cells were grown in 20610 cm dishes and
transfected with 60 nM miRNA mimic (Ambion/GenePharma)
using HiPerfect transfection reagent (Qiagen). 24 h later, cells
were suspended in ice-cold PBS by scraping and subjected to UV
crosslinking at 254 nM (Stratalinker). Cell pellets were lysed (0.1%
SDS, 0.5% deoxycholate, 0.5% NP-40 with protease inhibitors,
Roche) for 10 mins on ice followed by RQ1 DNAse (Promega) at
37uC for 15 mins with shaking. RNAse A/T1 (Ambion) was then
added for a further 8 mins, prior to the addition of EDTA
(30 mM). Pellets were then spun (30,000 rpm) and the lysate
subjected to immunoprecipitation for 2 h with a pan-anti-Ago
antibody (2A8, kind gift of Zissimos Mourelatos) conjugated to
protein-A dynabeads (Invitrogen) using bridging rabbit anti-mouse
IgG (Jackson Immunolabs). Pellets were then successively washed
(0.1% SDS, 0.5% deoxycholate, 0.5% NP40 in 16 PBS; 0.1%
SDS, 0.5% deoxycholate, 0.5% NP40 in 56 PBS; 50 mM Tris
pH 7.5, 10 mM MgCl2, 0.5% NP40) and on-bead phosphatase
Figure 1. Measurement of miRNA by qRT-PCR after transienttransfection with miRNA mimic. miR-200a, miR-125b and miR-16levels were quantitated by qPCR following either transfection of themiR-200a mimic in MDA-MB-231 cells, following addition of the miRNAmimic post lysis (doping), or after both transfection and doping.Experiments were performed as biological triplicates with error barsdepicting standard error of mean. Asterisks denote significance,*** p,0.001, ** p,0.01.doi:10.1371/journal.pone.0055214.g001
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 2 January 2013 | Volume 8 | Issue 1 | e55214
treatment performed for 30 mins with antarctic phosphatase (New
England Biolabs) in the presence of superasin RNAse inhibitor
(Ambion). The 39 RNA linker (CAGACGACGAGCGGG) was
labeled with P32 using T4-PNK (NEB) and ligated on-bead for 1 h
at 16uC with T4 RNA ligase (Fermentas). Beads were then washed
as previous and treated with PNK to ligate the 59 RNA linker
(AGGGAGGACGAUGCGGxxxG, with ‘‘X’’ representing differ-
ent nucleotides for barcoding). Beads were resuspended in 4x LDS
Novex loading buffer with 4% B-mercaptoethanol, incubated at
70uC for 10 mins and the supernatant loaded on Novex NuPAGE
4-12% Bis-Tris acrylamide gels (Biorad). After running, the Ago-
RNA complexes were then transferred to nitrocellulose and
exposed to film at 280uC for 3 days. Complexes running at
,110 kDa were then excised with a scalpel and resuspended
(100 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA, 4 mg/ml
proteinase K) for 20 mins at 37uC. The sample was incubated for
an additional 20 minutes in the presence of 3.5 M urea and RNA
isolated by a phenol-chloroform extraction. Sample was then run
on a 10% denaturing (1:19) polyacrylamide gel and exposed to
film with an intensifying screen at 280uC for 5 d. A thin band
corresponding to labelled miRNAs was excised, crushed and
eluted at 37uC for 1 h (1 M NaOAc, pH 5.2, 1 mM EDTA). RNA
was then precipitated overnight with ethanol, centrifuged and
dried. RNA was then resuspended in 8 ml H2O, primer added
(TCCCGCTCGTCGTCTG) and reverse transcription per-
formed using SuperScriptIII (Invitrogen). PCR was then per-
formed with the above primer and an additional primer
(ACGGAGGACGATGCGG) for 25 cycles. PCR product was
then run on a 10% native (1:29) polyacrylamide gel, stained with
Sybr Gold (Qiagen) and bands excised over a UV light box. The
DNA was then precipitated using isopropanol and a final 10 cycle
PCR performed with the following primers:
AATGATACGGCGACCACCGACTATGGATACTTAGT-
CAGGGAGGACGATGCGG, CAAGCAGAAGACGGCATA-
CGATCCCGCTCGTCGTCTG. Reactions were then run on
2% metaphor agarose/TBE gels and bands (,115 bp) excised
corresponding to the linker sequence + miRNA CLIP tag. Samples
were finally purified using quick-spin columns (Qiagen) and
subjected to Illumina GAII 35 bp read-length deep sequencing
(Geneworks).
Figure 2. Quantitation of functional transfected miRNA mimic by deep sequencing of RNA from Argonaute immunoprecipitation.A) MDA-MB-231 cells were transfected with miR-200a, with scrambled control, or were untransfected, then subjected to UV-crosslinking andArgonaute immunoprecipitation followed by deep sequencing of the Argonaute-bound small RNA pool. The levels of miRNAs (x-axis) are representedas a percentage of total miRNA sequencing reads (y-axis). Similar results were obtained using transfection of miR-200b. B) miR-200a and miR-125bwere measured by qPCR from whole cell lysate or by deep sequencing AGO-immunoprecipites from control and miR-200a-transfected MDA-MB-231cells. In each case the fold change is calculated by comparing to basal miR-200a levels.doi:10.1371/journal.pone.0055214.g002
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e55214
BioinformaticsUsing an in house Perl script, Illumina GAII 36 bp reads were
first filtered for average quality and for homopolymeric tracts
exceeding 12 nt, trimmed of linker sequence fragments and
separated by barcode. The program bowtie [12] was used to align
resulting 17 to 30 nt reads to the human genome (NCBI36/Hg18
downloaded from UCSC – http://genome.ucsc.edu), allowing a
maximum of one mismatch and permitting up to 15 map
locations. Reads with the same sequence were consolidated into
single BED format file entries, specifying the accumulated number
of tags and made available for display and analysis [12].
Fluorescent oligonucleotide transfection, Lysotrackertransfection, and immunofluorescence microscopy
MDA-MB-231 cells were transfected with 60 nM FAM labelled
negative control duplex oligonucleotide (Genepharma) on cham-
ber-slides coated with fibronectin and cultured in DMEM/20%
FCS. Following a 48 h incubation at 37uC, 5% CO2, 5 nM
Lysotracker red (Invitrogen) was applied to cells for 30 min. Cells
were washed in 37uC PBS for 5 min then fixed in 37uC 4% PFA,
pH 9.3 for 5 min. Cells were washed twice in PBS then blocked in
1% BSA/0.3% Triton X-100 for 20 min at RT. After three
washes in PBS cells were permeabilised in 0.3% Triton/PBS. Pan-
anti-Ago antibody (2A8, Zissimos Mourelatos) was applied at 1 in
250 for 1 h at RT. After 2x PBS washes, secondary antibody Alexa
Fluor 594 goat, anti-mouse IgG2a (Invitrogen, product No
Figure 3. Transfected siRNAs show little co-localisation with Argonaute. MDA-MB-231 cells were visualised by fluorescence microscopyshowing transfected fluorescent siRNA (green), endogenous Argonaute (red, visualised by immunofluorescence using 2A8 antibody) and nuclei(DAPI, blue). Cells were transfected using either A) Lipofectamine 2000 (Invitrogen) or B) HiPerfect (Qiagen). Representative images are shown.doi:10.1371/journal.pone.0055214.g003
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 4 January 2013 | Volume 8 | Issue 1 | e55214
Figure 4. Transfected siRNAs localise with and adjacent to lysosomes. MDA-MB-231 cells were visualised by fluorescence microscopyshowing transfected fluorescent siRNA (green), lysosomes (lysotracker, red) and nuclei (DAPI, blue). Cells were transfected with either A) HiPerfect(Qiagen) or B) RNAi-Max (Invitrogen).doi:10.1371/journal.pone.0055214.g004
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 5 January 2013 | Volume 8 | Issue 1 | e55214
A-11005) was applied for 1 h at RT. DAPI was applied for 5 min
then slides were washed twice in PBS and mounted in DAKO
aqueous fluorescent mounting medium. Cells were imaged using a
Leica SP5 spectral scanning confocal microscope.
miRNA inhibitor doping experiment60 nM antisense inhibitor miR-200a or scrambled control
antisense inhibitor (Dharmacon) was transfected into MCF7 cells
using Lipofectamine 2000 or Lipofectamine RNAi max (Invitro-
gen). Cells were incubated at 37uC, 5% CO2 for 48 h. Cells were
harvested with TRIzol reagent (Invitrogen). Prior to continuing
with RNA extraction samples were doped (added after cell lysis)
with the equivalent amount of miR-200a antisense inhibitor. qRT-
PCR was performed using Taqman probes (Applied Bioscience)
for miR-200a and miR-125b. Experiments were also performed
with 60 nM miR-200 or control LNA inhibitor (Integrated DNA
Technologies).
Acknowledgments
We would like to acknowledge David Lawrence for his assistance with
bioinformatics and members of the Goodall lab, especially Andrew Bert,
for useful discussion.
Author Contributions
Conceived and designed the experiments: DWT CPB GJG. Performed the
experiments: DWT CPB. Analyzed the data: DWT CPB JMS. Wrote the
paper: DWT CPB GJG.
References
1. Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and
siRNAs. Cell 136: 642–655.
2. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell
136: 215–233.
3. Karginov FV, Cheloufi S, Chong MM, Stark A, Smith AD, et al. (2010) Diverse
endonucleolytic cleavage sites in the mammalian transcriptome depend upon
microRNAs, Drosha, and additional nucleases. Mol Cell 38: 781–788.
4. Shin C, Nam JW, Farh KK, Chiang HR, Shkumatava A, et al. (2010)
Expanding the microRNA targeting code: functional sites with centered pairing.
Mol Cell 38: 789–802.
5. Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008) Endogenous siRNA
and miRNA targets identified by sequencing of the Arabidopsis degradome.
Curr Biol 18: 758–762.
6. German MA, Pillay M, Jeong DH, Hetawal A, Luo S, et al. (2008) Global
identification of microRNA-target RNA pairs by parallel analysis of RNA ends.
Nat Biotechnol 26: 941–946.
7. Bracken CP, Szubert JM, Mercer TR, Dinger ME, Thomson DW, et al. (2011)
Global analysis of the mammalian RNA degradome reveals widespread miRNA-
dependent and miRNA-independent endonucleolytic cleavage. Nucleic Acids
Res 39: 5658–5668.
8. Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, et al. (2005) Disruption of GW
bodies impairs mammalian RNA interference. Nat Cell Biol 7: 1267–1274.
9. Luo D, Saltzman WM (2000) Synthetic DNA delivery systems. Nat Biotechnol
18: 33–37.
10. Barreau C, Dutertre S, Paillard L, Osborne HB (2006) Liposome-mediated
RNA transfection should be used with caution. RNA 12: 1790–1793.
Figure 5. The passenger strand of an unmodified miRNA mimicis incorporated into RISC. MDA-MB-231 cells were transfected with60 nM scrambled or miR-200a (double-stranded) miRNA mimic.Samples were then subjected to Argonaute immunoprecipitation anddeep sequencing. Sequence read numbers are shown relative to miR-125b. A) Unmodified miRNA mimics showed significant co-precipitationof the passenger strand as well as the guide miR-200a strand. B) AmiRNA mimic modified to circumvent this problem (Ambion) success-fully eliminates incorporation of the passenger strand.doi:10.1371/journal.pone.0055214.g005
Figure 6. Measurement of miRNA by qPCR after antisenseinhibitor transfection does not reliably measure the level ofthe cellular miRNA. qPCR of miR-200a and miR-125b in MCF7 cellsfollowing transfection with antisense miR-200a (antisense inhibitor), orafter its addition post lysis (doping), or both. miR-200a antisenseinhibitors were either modified as A) 29-O-methyl oligonucleotides or B)LNA-oligonucleotides. Experiments were performed in biologicaltriplicate with error bars depicting standard error of mean. Asterisksdenote significance, *** p,0.001, ** p,0.01, * p,0.05.doi:10.1371/journal.pone.0055214.g006
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55214
11. Horwich MD, Zamore PD (2008) Design and delivery of antisense oligonucle-
otides to block microRNA function in cultured Drosophila and human cells. NatProtoc 3: 1537–1549.
12. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-
efficient alignment of short DNA sequences to the human genome. Genome Biol10: R25.
Pitfalls in Measuring miRNAs after Transfection
PLOS ONE | www.plosone.org 7 January 2013 | Volume 8 | Issue 1 | e55214